The role of clonal progression leading to the development of therapy-related myeloid neoplasms

Therapy-related myeloid neoplasms (t-MN) are characterized by aggressive features and a dismal prognosis. Recent evidence suggests a higher incidence of t-MN in individuals harboring clonal hematopoiesis of indeterminate potential (CHIP). In order to gain insight into CHIP-driven malignant progression, we gathered data from ten published reports with available detailed patient characteristics at the time of primary malignancy and t-MN development. Detailed clinical and molecular information on primary malignancy and t-MN were available for 109 patients: 43% harbored at least one somatic mutation at the time of the primary malignancy. TET2 and TP53 mutations showed an increasing variant allele frequency from CHIP to t-MN. ASXL1-associated CHIP significantly correlated with the emergence of TET2 and CEBPA mutations at t-MN, as well as U2AF1-driven CHIP with EZH2 mutation and both IDH2 and SRSF2-driven CHIP with FLT3 mutation. DNMT3A-driven CHIP correlated with a lower incidence of TP53 mutation at t-MN. In contrast, TP53-driven CHIP correlated with a complex karyotype and a lower tendency to acquire new mutations at t-MN. Patients with multiple myeloma as their first malignancy presented a significantly higher rate of TP53 mutations at t-MN. The progression from CHIP to t-MN shows different scenarios depending on the genes involved. A deeper knowledge of CHIP progression mechanisms will allow a more reliable definition of t-MN risk. Supplementary Information The online version contains supplementary material available at 10.1007/s00277-024-05803-y.


Introduction
Therapy-related myeloid neoplasms (t-MN) or myeloid neoplasm post cytotoxic therapy (MN-pCT), as defined according to the 5th edition of WHO Myeloid Neoplasia Classification, encompasses conditions such as acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) with a documented history of chemo/ radiotherapy as treatment for an unrelated condition [1,2].Although "post cytotoxic therapy" or "therapy-related" (as per International Consensus Classification, ICC, nomenclature [3]) have lost their autonomous entity status and are disease attributes in the new classifications, the recognition of this feature remains of major importance.
t-MN are characterized by poor prognosis and a specific karyotype/genetic signature, enriched in complex cytogenetic abnormalities and high-risk mutations, especially TP53, detected in approximately 20-40% of cases [4].The precise mechanisms leading to malignancy development, involving complex microenvironment interactions, inherited predisposition, drug genotoxicity and clonal selection, are still the object of extensive study [4,5].Clonal hematopoiesis of indeterminate potential (CHIP) has been considered one of the main risk factors and has been identified at the time of the primary cancer diagnosis in 30-70% of patients developing a t-MN, potentially being the primum movens of clonal evolution toward malignancy [6][7][8][9].Most individuals with CHIP, defined as the presence of mutations in myeloid genes at a variant allele frequency (VAF) ≥ 2% [10], exhibit somatic mutations in genes responsible for epigenetic regulation, such as DNMT3A, TET2 and ASXL1, while only a minority have mutations in splicing genes like SF3B1, SRSF2 and U2AF1, and TP53, whose prevalence range from 0.03 to 0.2% [11,12].
The worldwide diffusion of next-generation sequencing (NGS) techniques has allowed the identification of somatic mutations at very low VAF in t-MN patients, emphasizing the concept of clonal evolution.However, characterizing the role of somatic mutations in t-MN patients remains challenging because of the limited sample sizes and the difficulties in collecting paired samples at primary diagnosis and t-MN, from patients who share the same primary tumor and treatment history.
In a recently-published paper, we investigated the prevalence of CHIP in a cohort of patients who developed a t-MN after treatment with chemo(immuno)therapy [13].
In the present study, we aim to expand our observations by conducting a review of data available in the literature in order to characterize the role of specific gene variants in t-MN development and to gain novel insights on malignant progression of individuals with CHIP exposed to cytotoxic treatments.Knowledge of the mechanisms underlying progression of CHIP will allow for better t-MN risk stratification and the development of patient-specific treatments.

Patient characteristics
This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statements [14].The literature search focused on studies reporting t-MNs with available detailed genetic information (including at least the 29 genes selected for the purpose of this study, see Supplementary Table 1) at the time of primary malignancy and at the time of t-MN.The systematic search retrieved 146 patients from 15 studies [6,9,13,[15][16][17][18][19][20][21][22][23][24][25][26]; after the eligibility check, 109 patients from 10 studies were selected [6,13,16,17,[19][20][21][22]24].The PRISMA flow chart of the selection procedure and main reasons for exclusion are detailed in Fig. 1 (More details in supplementary material).Supplementary tables 2 and 3 show the studies included in the analysis, the gene panel selected for the analysis, the time of genetic screening and the variants identified.The nature of variants whose pathogenicity was not clearly stated in the original papers was assessed and clearly marked with asterisks (supplementary Table 3).We identified only 6 variants of uncertain significance (VUS), that for consistency with original papers were included in the analysis.
When available, the following data were collected: age at the time of primary tumor (t0), sex, type of primary malignancy, treatment received (including autologous stem cells transplant [ASCT]), type of t-MN, latency between primary malignancy and t-MN, genetic mutation at t0 and t-MN, source of genetic analysis at t0 and t-MN, karyotype analysis at t0 (none of the studies included presented this data) and t-MN (see Table 1).The patients were considered to have CHIP if the genetic mutations presented VAF ≥ 2%.To evaluate the mutational burden changes between t0 and t-MN, we identified a 10% cut-off to define a significant increased/decreased VAF.TP53 mutations were considered multi-hit according to both WHO-5 and ICC classifications [3,27].Other mutations were considered to be multi-hit if either 2 different mutations on the same gene or a single mutation with VAF > 50%.Complex karyotype was defined by the presence of more than or equal to 3 aberrations.

Statistical analysis
Univariate and multivariate analyses were used to establish associations between the variables.Categorical variables were compared using the chi-square test; odds ratios (OR) with 95% confidence intervals (CI:95%) were also calculated (unless one of the cells of the contingency table for the categorical variables is equal to 0).For expected cell values less than 5, Fisher's exact test and the exact limits for confidence intervals were preferred.The Mann-Whitney test was used for continuous variables as appropriate.A p-value less than 0.05 was considered as statistically significant.Statistical analysis was performed through IBM SPSS Statistics 27 (IBM Corp. in Armonk, NY).

Demographic characteristics
One hundred and nine patients were included in this study (Median age 62 yr [range 18-79]; M/F ratio 1.5).The more frequent primary tumors were lymphoid malignancies (58%), solid tumors (26%) and plasma-cell dyscrasias (Multiple myeloma or AL amyloidosis, 16%).Data on previous therapies were available in 71 patients: 21 underwent chemotherapy and 50 a chemo-radiotherapy combination; ASCT was carried out in 47% of patients.The most common t-MN was MDS (61%), while AML was diagnosed in 38% of cases and the type of t-MN was not specified in 16% of patients.The median latency between t0 and t-MN was 36 months (range 2-183) (Table 1).
The most common mutations at t0 were TP53 (16%), TET2 (12%), DNMT3A (12%) and ASXL1 (4%) (Fig. 2).For all genes, there was an increase of VAF between t0 and t-MN, which was significant only for TET2 (median VAF t0: 3.5% vs t-MN: 21.2%, p = 0.019) and TP53 (median VAF t0: 5% vs t-MN: 31.95%,p = 0.005).Of note, in patients with TP53 mutations at t0, both the INDEL mutations (n = 2) showed a VAF increasing over time, whereas missense mutations expanded in half of the cases (6 out of 12), but the difference did not reach statistical significance, probably due to the limited number of patients with INDEL mutations.In patients with TP53-driven CHIP with a stable VAF (variation ≤ 10%), the TP53 Mut clone at t-MN was not detectable in two cases (UPN 56 and 103 in supplementary Table 3).The first patient developed a t-MDS characterized by a single GATA2 mutation (VAF: 33%), and inv(3) three years after treatment with Temozolomide and radiotherapy for Glioblastoma.The patient was treated with Fludarabine, Idarubicin and Cytarabine for the t-MN and progressed successfully to hematopoietic stem cell transplantation [6].The second patient developed a t-MDS (subsequently evolved to t-AML) with complex karyotype, without genetic lesions after multiple chemo-radiotreatments for lymphoma [26].No additional differences were detected between type of mutation and VAF changes (supplementary Fig. 2).Most frequently mutated genes at t-MN were TP53 (45%), DNMT3A (20%), TET2 (15%) and NRAS (14%); all of these variants but TET2 were more frequent when compared with t0 (p < 0.0001, p = 0.004, p = 0.375 and p = 0.003, respectively; Fig. 2).The most common mutations emerging at t-MN or not detectable at t0 were TP53 (35%), NRAS (9%), RUNX1 (9%) and DNMT3A (9%), although we cannot exclude that these mutations were present at values below the detection threshold declared by the study.In particular, TP53 and RUNX1 genes presented a high rate of multi-hit mutations (63% and 60%, respectively).A comprehensive representation of the dynamic mutational profile of the patients with information on primary tumor and ASCT is shown in Fig. 3.

Focus on main CHIP driver mutations
Given the well-known role of the TP53 mutation in t-MN pathogenesis, we then investigated this mutation: patients who were TP53 wt at t0 developed no new mutations at t-MN diagnosis in 29% of cases, 1 new mutation in 29% of cases and 2 new mutations in 25% of cases.In contrast, patients who carring TP53 mutation at t0 were more likely to have no new mutations at t-MN (13 patients, 81%), with a few developing a high number of new mutations (3 mutations in 2 patients, 12.5% and 7 mutations in 1 patient, 7%; Fig. 5).
Overall, the presence of TP53 mutation at t0 was associated with a lower incidence of new mutations at t-MN (p < 0.001, OR 0.094, 95%CI 0.025-0.358).Furthermore, both the emergence of a new TP53 mutation at t-MN and the presence of TP53 mutation at t-MN correlated with a primary plasma-cell dyscrasia (p = 0.001 and p = 0.039, respectively; Fig. 5).Information about the treatment of patients with this type of primary tumor were available for only 5 patients.The only two patients undergoing treatment with Lenalidomide developed TP53 mutations at t-MN not detected at t0 (in both cases the sensitivity reported for the NGS method was 1% VAF) [21].
As expected from previous reports, the other most frequently mutated genes at t0 were TET2 (15 mutations in 13 patients), DNMT3A (14 mutation in 13 patients) and ASXL1 (4 mutations in 4 patients).

Discussion
The comparative analysis of genetic features of samples in patients at the time of first malignancy and at the time of developing t-MN allowed us to observe a significant higher frequency of genetic variants and cytogenetic abnormalities in the second time point, as expected, an increasing VAF of specific hits between t0 and t-MN, such as TET2 and TP53, possibly indicating a main role in leukemogenesis, and a lower incidence of TP53 mutation at t-MN in patients harbouring a DNMT3A-driven CHIP.
To gain insight into malignant progression of individuals harboring CHIP mutations undergoing cytotoxic treatments, we compared the genetic landscape of somatic mutations at t0 of the cohort of patients we gathered from literature to the one found in the non-hematological patients (NHP) as reported in the seminal manuscripts by Jaiswal et al. and Genovese et al. [12,28].The first difference that stands out is the higher rate of patients with CHIP in our cohort (43% vs 5% in age-matched individuals [60-65 yrs.]).It should be pointed out, though, that part of the samples at t0 were collected before t-MN onset but after some cycles of chemotherapy (e.g. at the time of hematopoietic stem cell [HSC] collection for ASCT), which could trigger a clone selection, and the possibility that mutations identified as CHIP drivers could have been found in clones responsible for the primary malignancies.
When compared to NHP, the patient cohort showed underrepresentation of DNMT3A (the most common mutation in NHP), ASXL1 (whose mutation frequency in NHP is similar to TET2), JAK2, SF3B1 and CBL mutations, with overrepresentation of TP53 and TET2 mutations.The importance in malignant progression of TP53-and TET2-driven CHIP is also confirmed by the significant VAF increase from t0 to t-MN.
As expected from previous reports, the cohort of t-MN selected for this study presented high rate of cytogenetic abnormalities, in particular del (5), del (7) and complex karyotype, and characteristic genetic signatures, with low incidence of mutations commonly detected in de-novo AML, such as NPM1 and FLT3, and higher incidence of TP53, NRAS and KRAS mutations [4].
Recent reports pointed out that NK t-MN is an entity clinically and genetically distinct from classical t-MN harboring karyotype abnormalities.NK t-MN present better prognosis, lower frequency of TP53 mutations and higher frequency of TET2, NPM1, ASXL1, SRSF2, RUNX1, KRAS, FLT3 and STAG2 mutations [29][30][31].Our small cohort of NK t-MN is in line with some of the mutational features previously reported, as enrichment in TET2 and KRAS mutations.Intriguingly, we detected a higher, although not statistically significant rate of CHIP at t0 when compared to non-NK t-MN (71% vs 42%).If confirmed in a larger cohort of patients, this difference could let us envision a different preferential mechanism of progression between the two entities: one CHIP-linked (through clone expansion and/or acquisition of additional mutations), more common in NK t-MN, versus correlated to chromosomal destabilization, specific for abnormal karyotype t-MN, characterized by TP53 mutations at the time of first malignancy.The lack of cytogenetic analysis at t0 does not allow us to further speculate on the last mechanism, which could be fostered by karyotype abnormalities or single nucleotide polymorphisms before the onset of t-MN or be the consequence of the exposure to genotoxic agents over the TP53 mut karyotypic ground [4].
Current evidence on the mechanisms of progression of CHIP to MN highlight similarities and differences among specific gene-driven CHIPs.Among these, DNMT3A, TET2 and ASXL1 mutations are often detected at considerably higher VAF compared to NRAS, KRAS and GNAS [32,33].Analyzing a large cohort of elderly individuals, Van Zeventer et al., highlighted that JAK2, TET2, ASXL1 and TP53 somatic lesions, but not DNMT3A, tend to increase their allelic burden over time.Furthermore, TET2 and ASXL1mutated diseases present a higher propensity to acquire additional mutations over time, as compared to those carrying DNMT3A mutations [34].Intriguingly, in the studied cohort, patients harboring TET2 mutations at t0 did not show a different pattern of emerging mutations at t-MN, when compared to other patients; instead, TET2 VAF significantly increased between t0 and t-MN.These findings lead to the hypothesis that TET2 mut clones could preferentially expand and not accumulate mutations, when exposed to cytotoxic therapy.
Several papers have focused on the leukemogenesis process of TP53 mut MN, recently recognized by ICC as an autonomous category due to the high-risk features and adverse outcomes [3,35,36].In particular, in-vitro and invivo studies showed that cytotoxic therapy selects low-VAF TP53 mut clones which present survival advantage preferentially expanded after treatment [37].In the same line, Bolton et al. documented a strong association between TP53-driven CH and previous exposure to cancer therapy [38].Accordingly, Shah et al., analyzing a cohort of nearly five hundred t-MN patients, found a high percentage of TP53 mutations (37%).Strikingly, the total number of co-mutations was significantly inferior in TP53 mut cases compared to TP53 wt , with more then 60% of TP53 mut patients not presenting comutations [39].The small number of co-mutations detected Fig. 3 Mutational profile of the patients, grouped for type of primary tumor, with information on autologous stem cell transplant (ASCT).Each row refers to a single patient.The color of the box represents the changes in the VAF of the mutations between t0 and t-MN as indicated by the legend.NA: Not available.VAF: variant allele frequency ◂ by Shah et al. and confirmed in our literature review is consistent with the high rate of TP53 biallelic mutations, feature found to worsen prognosis and linked to a smaller co-mutational burden when compared to monoallelic mutations [40,41].
Taken together, this evidence highlights a discrepancy between the rate of cytogenetic abnormalities and the gene co-mutational burden in TP53 mut t-MN.Accordingly, several authors demonstrated the key role of TP53 in maintaining diploid karyotype [42][43][44]; more precisely, TP53 induces apoptosis in cells that have a long pause in the mitotic checkpoint, which indicates DNA damage [45].
Taking advantage of CRISPR-Cas9 technology, Boettcher et al. demonstrated a dominant negative effect of TP53 missense mutations, which would confer similar drug resistance and survival advantage when compared to TP53 ko models [46].Intriguingly, in our patients gathered from the literature, the two patients carrying a truncating TP53mutation at t0 showed increased VAF at t-MN, whereas among the 12 patients with a missense mutation, only half of them presented an increased VAF.Although not statistically significant, probably due to the low number of studied patients, this association could point out differences in therapy-related risk progression in different TP53 mutations.Unfortunately, the small number of patients does not allow definitive conclusions and underlines the need to extend the study of the role of CHIP to larger cohorts of patients who develop t-MN following treatment with chemo-and/or radio-therapy.
Recently, Sperling et al. demonstrated, in in vitro and in vivo models, that lenalidomide, but not pomalidomide, provides a selective advantage to TP53 mut HSCs [47].Similarly, in the present case series, we documented the emergence of TP53 mutations in the two patients exposed to lenalidomide.Accordingly, it is worth highlighting that clonal hematopoiesis at a VAF below 2% (age-related clonal hematopoiesis, ARCH), was not included in our study, due to inconsistencies in the limit of resolution of selected papers, but could be more frequent than CHIP, as shown in NHP by previous reports [48,49].Thus, it is plausible to envision a larger rate of patients harboring clonal hematopoiesis at the time of primary malignancy; accordingly, several mutations emerging at t-MN (especially TP53) could be present also before the cytotoxic therapy and thus be selected thanks to the resistance to treatments.
In sum, the comparative analysis of molecular profiles pre-and post-t-MN onset allowed us to gain interesting insights on malignant progression in CHIP-carrying individuals, following exposure to cytotoxic treatments.
Our analysis of the data from both the perspective of CHIP-driven mutations and mutational landscape of t-MNs, corroborates the findings by previous authors and suggest a potential leukemogenic role of both TET2 and TP53-driven CHIP.Although previous reports have showed an increasing of the TET2 clones over time and in condition of aging/ inflammation [34,50], this is the first systematic analysis showing their potential role in the process of therapy-related leukemogenesis.Furthermore, we highlighted a preferential leukemogeneis process by TP53-driven CHIP, based on clone expansion rather than mutations acquisition.This feature resulted to be more common in clones harbouring INDEL TP53 mutations, which, thus, could present an even higher risk of expansion when compared to missense mutations.
Intriguingly, patients carrying DNMT3A mutations at t0 presented a lower incidence of TP53 variants at t-MN.This observation lines up with the lower trend to increase VAF over time and to acquire new mutations in DNMT3A-driven CHIP, both in patients developing t-MN and in healthy individuals, as shown by Van Zeventer et al. [34], and a lower risk of myeloid malignancy when compared to other genotypes [50].In sum, these findings suggest that, when compared to other CHIP-drivers, DNMT3A presents lower leukemogenic potential and may eventually cause a milder disease phenotypes.
Unfortunately, the paucity of the data available did not allow further analyses to investigate specific therapyrelated effects (e.g.ASCT or specific drug-based treatments) and to study more in deep gene-specific mutational  5 patterns.In this regard, another limitation of our study is the exclusion of PPM1D from most gene panels, whose mutations are a known hematopoietic driver in response to DNA damage [51].
In this perspective, thanks also to the availability of biobanks in cancer centers, broader, comparative, prospective studies are warranted to further investigate CHIP progression mechanisms that could allow, in the near future, a better stratification of the individual risk of developing a t-MN.In this line, availability of alternative, and possibly targeted treatments, may better direct the therapeutic approach to hematologic malignancies and solid tumors, with the goal of reducing the rate of this unfavorable complication.
need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http:// creat iveco mmons.org/ licen ses/ by/4.0/.

Fig. 1
Fig. 1 PRISMA flow diagram of patients enrollment

Fig. 2
Fig. 2 Mutational landscape in the patient population.2A) Number of mutations at the time of diagnosis of first malignancy (t0) and at t-MN development 2B Landscape of mutations emerged at t-MN.

Fig. 4
Fig. 4 Correlation between mutations emerged at t-MN.The figure shows the correlations between mutations identified at t-MN but not present at t0. Asterisks indicate statistically significant correlations (p < 0.05).The strength of the correlation is indicated in color shades as shown by the legend below the figure.Only mutations occurring at least once at t-MN are shown.For further information please refer to supplementary Table 5